A flow cytometer is used for studying particles, such as cells and latex beads, which are suspended in a liquid stream that is passed through an imaging apparatus for a prolonged time. Recently, an imaging flow cytometer technology, termed ImageStream™, has been developed for evaluating cells and other that are conveyed through an imaging apparatus. These significant advancements in the art of flow cytometry are described in commonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001 and entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955 issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as commonly assigned U.S. Pat. No. 6,473,176, issued on Oct. 29, 2002, and also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawings of each of these patents are hereby specifically incorporated herein by reference.
In a manner similar to a conventional flow cytometer, an ImageStream™ imaging flow cytometer uses hydrodynamic focusing to confine a sample core fluid containing cells of interest to a central portion of a flowing stream of a cell-free sheath fluid. Sheath flow improves the precision with which the cell sample can be positioned in an observation region of the cytometer by restricting the cells to the central region of the stream. An additional advantage of using sheath flow is that it reduces the possibility of clogging in the fluidic system. By using sheath flow, a typical core flow diameter of about 10 microns and a sheath flow diameter of about 200 microns are achieved. Without the use of sheath flow, the flow cell would need to be restricted to about 10 microns in diameter, thus allowing particles of about 10 microns or greater in size to obstruct the fluidic system.
Currently, most flow cytometers employ gas pressure to inject the core and sheath streams. The gas pressure injection is accomplished by connecting a sample vial containing the liquid sample to the fluidic system via an o-ring seal, and inserting two fluid lines into the sample vial. The first fluid line is a gas pressure fluid line, which is generally coupled in fluid communication with the head space of the sample vial. The second fluid line is disposed adjacent the bottom of the sample vial. As the pressure is increased in the head space of the sample vial, the liquid sample is forced into the second fluid line, so that the liquid sample is removed from the sample vial.
Such a conventional pressurized gas injection technique is inadequate for the recently developed ImageStream™ imaging flow cytometer for at least two reasons. First, the low flow rates used in the ImageStream™ system would require very low gas pressures, which are quite challenging to regulate accurately. Second, such gas systems are constant pressure systems, which are prone to clogging. A more preferred injection system employs constant volume pumping, such as provided by syringe pumps.
One example of a syringe pump is disclosed in U.S. Pat. No. 5,176,646 (Kuroda), which describes a single speed syringe pump using a stepping motor and implemented with a plurality of belts and pulleys, providing a low cost alternative to more expensive syringe pumps that include precision machined lead screws. For example, U.S. Pat. No. 5,219,099 (Spence et al.) discloses a syringe pump that uses a stepping motor to rotate a lead screw with precision. The syringe barrel, plunger, and stepper motor drive shaft are all coaxially aligned with the syringe barrel axis in order to eliminate all forces that are not coaxially aligned.
However, existing syringe pumps suffer from pulsatility issues and a general inability to offer both precisely controlled delivery of small volumes of fluid, as well as the inability to rapidly reload the fluid reservoir of the syringe. Pulsatility refers to the introduction of rhythmic or irregular pulses into a flow of fluid discharged by a fluid delivery system. Particularly with respect to the delivery of small volumes of fluid, such pulsatility is undesirable. It would therefore be desirable to provide new method and apparatus for the delivery of small volumes of fluid to enable the fluid to be delivered at a constant volume with low pulsatility, while enabling a uniform suspension of cells, beads, or other particles of interest in the sample fluid, and enabling the rapid reloading of the source syringe—all without sacrificing the ability to accurately deliver small volumes of fluid.
The pulsatility issue is particularly important in an imaging flow cytometer. The ImageStream™ optical imaging flow cytometer system, as described in commonly owned U.S. Pat. No. 6,249,341 (Basiji et al.) and U.S. Pat. No. 6,211,955 (Basiji et al.) provides high resolution, high sensitivity two-dimensional (2D) and three-dimensional (3D) imaging using time-delay-integration (TDI) electronic image acquisition of cells in flow. These instruments are designed to expand the analysis capabilities of biological specimens in fluid suspensions beyond the limits of conventional flow cytometers. TDI sensors use solid-state photon detectors, such as charge-coupled device (CCD) arrays and shift lines of photon-induced charge in the arrays in synchronization with the flow of the particle of interest. The method enables a long exposure time that increases the signal-to-noise ratio (SNR) in the image, while minimizing blurring. Precise synchronization of the TDI detector timing with the motion of the moving targets is required for blurring of the resulting images to be eliminated or minimized. For example, if a target traverses 512 lines of a TDI sensor to build an image, and blurring of less than a single line width is desired, the velocity of the target must be known with a maximum permissible error of less than 0.2 percent.
To synchronize the TDI detector read out rate with the sample, a novel velocity detection method and apparatus has been developed. Commonly assigned U.S. patent application Ser. Nos. 09/939,292 and 09/939,049, both entitled MEASURING THE VELOCITY OF SMALL MOVING OBJECTS SUCH AS CELLS, describe a method and apparatus for determining a particle's velocity with a high degree of precision. These specification and drawings of both these applications are hereby specifically incorporated herein by reference. Light from an object entrained in the sample liquid core of the flow cell is modulated via an optical grating. The modulated light is detected and analyzed either in the time or frequency domain to determine the velocity of the object.
The velocity detection and synchronization method and apparatus disclosed in the above-identified patent applications are preferably implemented at a rate of approximately five times per second, enabling the determination of velocity to within 1 part in 1000. Such an implementation is adequate for relatively low frequency changes in cell flow speed (i.e., <5 Hz); however, it is not adequate for high frequency oscillations in cell velocity (i.e., where pulsatility in fluid flow exists). While low frequency changes in flow velocity can be compensated by using the velocity detection and synchronization method and apparatus disclosed in the above-identified patent applications, such velocity variations effectively decrease the maximum throughput flow velocity of the system, since the system must operate at speeds lower or equal to the maximum TDI detector read-out rate. Therefore, if the system has a high variation in low frequency velocity, the average velocity must be decreased so as not to surpass the TDI detector read-out rate. Therefore, it would be desirable to provide a fluidic pump that has a low degree of pulsatility.
In a current implementation of the aforementioned ImageStream™ system, sample analysis rates range from about 100 to about 5,000 cells per second. The maximum rate thus achieved is not as high as is employed in conventional flow cytometers, which often have sample rates ranging from about 10,000 to about 100,000 cells per second. Moreover, because of the use of a TDI detector in the aforementioned ImageStream™ system, the analysis time for samples analyzed in an ImageStream™ system is actually much longer than in conventional cytometers. Analysis times of up to 20 minutes or longer, which can provide greater accuracy, can be achieved using an ImageStream™ system. Given such lengthy analysis times, it is likely that if the sample were not mixed or agitated occasionally, the concentration of the sample fluid would not be uniform (i.e., some particles or cells would settle towards the bottom of the sample vessel). Such settling is undesirable, and would result in fluidic sample streams that were not representative of the particle concentration of the original sample being analyzed. Furthermore, in some assays, the concentration of particles can be chosen in order to optimize the analysis rate (cells/second), and such settling would interfere with the optimization. Also, some samples will likely include mixtures of cells or particles having different densities (e.g., whole blood includes different types of cells having different densities), and the different densities would result in some cells or particles settling out more rapidly than others, thereby undesirably distorting the analytical results achieved.
Particles and cells entrained in a fluid become distributed in a non uniform fashion due to the effect of gravity on the particles and cells. Research into the effects of gravity in such cases has resulted in the development of a technique for simulating the effect of microgravity on fluids. In the simulation, a sample container is rotated about its axis, so that the contents of the sample container are exposed to a uniformly varying simulated gravitational field, as opposed to a stationary gravitational field. U.S. Pat. No. 5,104,802 (Rhodes et al.) describes a clinostat for simulating microgravity. In this patent, cells are introduced into a hollow fiber, and the ends of the fiber are sealed with wax. This fiber is porous and is placed into a glass tube that contains a culture media. The fiber and tube are rotated about the longitudinal axis of the tube, which is horizontal, to simulate microgravity during cellular growth.
Rotation of a fluid has also been employed to minimize convective effects in the fluid. U.S. Pat. No. 4,040,940 (Bier) discloses an electrophoretic separation apparatus for soluble or particulate ionized matter. A tube is rotated about its longitudinal axis, while horizontal, to effectively continuously change the gravity vector, which minimizes gross convective effects that can interfere with the electrophoretic separation process. An anode and cathode are placed on separate ends of the separation tube, and a buffer is introduced on the cathode side of the tube while a perpendicular elution stream is provided to enable the collection of separation fractions.
U.S. Pat. No. 5,026,650 (Schwarz et al.) discloses rotating a horizontally disposed cylindrical cell culture vessel for optimizing cell growth. Delicate mammalian cells are uniformly suspended via the rotating culture vessel without turbulent action which can harm the cells. Other similar rotating vessels for cell culture include U.S. Pat. No. 5,155,035 (Schwarz et al.), U.S. Pat. No. 5,155,034 (Wolf et al.), U.S. Pat. No. 5,153,133 (Schwarz et al.), U.S. Pat. No. 5,153,131 (Wolf et al.), U.S. Pat. No. 4,988,623 (Schwarz et al.), U.S. Pat. No. 5,153,132 (Goodwin et al.), and U.S. Pat. No. 6,080,581 (Anderson et al). The disclosure and drawings of these patents are hereby also specifically incorporated herein by reference. A paper entitled “Particle Orbits in a Rotating Liquid” by William W. Fowlis and Dale M. Kornfeld published in the Journal of Fluid Mechanics, 1991 describes the difficulty experienced by researchers in producing latex microspheres greater than three microns. Such particles either settled or creamed/floated to the surface as a result of their buoyancy because they are lower in density than the surrounding medium. Prior art agitation devices such as paddle or propeller-type stirrers resulted in aggregation or flocculation of particles during the latex bead polymerization reaction. In light of these difficulties, such large diameter particles were only successfully manufactured in space, in experiments performed on the U.S. Space Shuttle, since the micro-gravity environment provided there enabled the beads to be suspended uniformly during polymerization.
An earth-bound rotary reactor was later developed to achieve a similar suspension in a gravity environment. By slowly rotating the vessel, the fluid contents begin to turn as a solid body, due to drag applied by the cylinder wall. The sedimentation or buoyancy of the particles is countered by the continuously changing gravity vector, causing the particles to trace a circular orbit with centers displaced from the axis of rotation. The above-referenced paper by Fowlis and Kornfeld calculates the optimal rotation rate as a function of the many variables, including particle and fluid densities, Reynolds and Taylor numbers associated with the fluid, and the vessel diameter.
U.S. Pat. No. 5,330,908 (Spaulding) discloses a culture vessel for growing mammalian cells constructed in a one piece integral configuration with an opening that is closed by an end cap. This culture vessel is rotatable horizontally about its longitudinal axis using a conventional roller system. The open end has a tapered access port to receive the ends of a hypodermic syringe. Such syringes enable the introduction of fresh nutrient and the withdrawal of spent nutrients.
U.S. Pat. No. 6,387,077 (Klibanov et al.) discloses a syringe pump that suspends particles as the syringe barrel is either intermittently or continuously revolved in a planetary orbit about an axis of rotation that is parallel to the syringe barrel axis. This apparatus is used to introduce biomedical contrast imaging agent into the vascular system of a patient for imaging tissue or organs. In this application, it is advantageous for the agent to be uniformly suspended and injected to achieve improved image quality. In an alternative configuration, a motor is used to both inject and rotate a syringe barrel inside an outer cylindrical housing. While the syringe pump disclosed in U.S. Pat. No. 6,387,077 enables the contents of a syringe to be agitated, the syringe pump does not provide either the desired low pulsatility or rapid reloading capability. It would thus be desirable to provide a syringe pump offering delivery with low pulsatility, which can be rapidly reloaded, and which enables a uniform suspension of cells, beads, or other particles of interest in a sample fluid.